Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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In Vivo Wound Healing Activity of Crocodile (Crocodylus siamensis) Hemoglobin and Evaluation of Antibacterial and Antioxidant Properties of Hemoglobin and Hemoglobin Hydrolysate

  • Pakdeesuwan, Anawat (Department of Biochemistry, Faculty of Science, Khon Kaen University) ;
  • Araki, Tomohiro (Department of Bioscience, School of Agriculture, Tokai University) ;
  • Daduang, Sakda (The Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Faculty of Science, Khon Kaen University) ;
  • Payoungkiattikun, Wisarut (The Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Faculty of Science, Khon Kaen University) ;
  • Jangpromma, Nisachon (The Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Faculty of Science, Khon Kaen University) ;
  • Klaynongsruang, Sompong (Department of Biochemistry, Faculty of Science, Khon Kaen University)
  • Received : 2016.03.21
  • Accepted : 2016.09.26
  • Published : 2017.01.28
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Abstract

The hydrolysis of proteins constitutes an invaluable tool, granting access to a variety of peptide fragments with potentially interesting biological properties. Therefore, a hemoglobin (Hb) hydrolysate of Crocodylus siamensis was generated by digestion under acidic conditions. The antibacterial and antioxidant activities of the Hb hydrolysate were assessed in comparison with intact Hb. A disc diffusion assay revealed that the Hb hydrolysate exhibited antibacterial activity against eight strains of gram-positive bacteria and showed a higher efficacy than intact Hb. Moreover, the antioxidant activity of intact Hb and its hydrolysate was evaluated using ABTS and DPPH radical scavenging assays. The Hb hydrolysate exhibited free radical scavenging rates of 6-32%, whereas intact Hb showed a slightly higher activity. In addition, non-toxicity to human erythrocytes was observed after treatment with quantities of Hb hydrolysate up to $10{\mu}g$. Moreover, active fragmented Hb (P3) was obtained after purifying the Hb hydrolysate by reversed-phase HPLC. Scanning electron microscopy demonstrated the induction of bacterial cell membrane abnormalities after exposure to P3. Antibacterial and antioxidant activities play crucial roles for supporting the wound healing activity. Consequently, an in vivo mice excisional skin wound healing assay was carried out to investigate the effects of intact Hb treatment on wound healing in more detail. The results clearly demonstrate that intact Hb is capable of promoting 75% wound closure within 6 days. These findings imply that intact Hb of C. siamensis and its acid hydrolysate may serve as valuable precursors for food supplementary products benefitting human health.

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